The high energy demand of neural processing must be matched by the supply of nutrients from the blood. In a process called functional hyperemia, energy demands are met by linking neural activity with dilation of penetrating arterioles, vessels that deliver blood to the brain. It is critical to understand the cellular mechanisms underlying functional hyperemia because it is the physiological basis for a widely-used research tool named blood-oxygen-level dependent (BOLD) contrast imaging, and because impaired functional hyperemia is implicated in the pathogenesis of Alzheimer's disease, stroke, and other devastating neurological diseases. The goal of this project is to investigate a novel cellular mechanism of functional hyperemia involving pericytes, enigmatic cells embedded within capillary walls. By definition, functional hyperemia begins with neural activity and ends with vascular smooth muscle cell (VSMC) hyperpolarization, which causes penetrating arteriole dilation and increased blood flow. However, the intermediary steps of functional hyperemia are unclear. Pericytes are ideally-positioned to play a role in the intermediary steps of functional hyperemia because they are in close proximity to neurons, they respond to neurotransmitters ex vivo, and they are structurally and functionally connected to VSMCs. Although pericytes may not have the machinery to change blood flow directly, they are capable of rapidly communicating changes in membrane potential and cytosolic calcium to VSMCs. Considering these findings, we are intrigued by the possibility that pericytes transduce neural activity into vasodilation by communicating with VSMCs. Here we propose to examine this novel route of neural- vascular communication by testing the hypothesis that pericytes respond to neural activity, and do so before VSMCs. In two aims, we will test this hypothesis using in vivo two-photon imaging of novel transgenic mice expressing calcium sensors in pericytes and VSMCs. In Aim 1, we will measure sensory stimulation- evoked changes in pericyte and VSMC calcium concentration. In Aim 2, we will optogenetically stimulate pyramidal neurons and observe pericyte and VSMC calcium changes. Preliminary data under physiological conditions show that pericyte and VSMC calcium levels are significantly positively correlated with each other, and negatively correlated with arteriole diameter. Moreover, pericytes and VSMCs exhibited a significant sensory stimulation-evoked calcium decrease. These findings support our hypothesis and the existence of a novel neuron-pericyte-VSMC communication axis. To more precisely correlate neural activity with vessel calcium, and to unravel which neurons may communicate with the vasculature, we will optogenetically stimulate pyramidal neurons while imaging pericyte and VSMC calcium and vessel lumen diameter. The use of novel mouse models and imaging techniques to elucidate new cellular interactions represents an outstanding training opportunity that may help to answer important questions in the field.